Peptides are a rapidly growing class of therapeutics with more than 50 peptide-based products currently on the market and even more in development covering disease areas such as immunology, oncology, neurology and endocrinology. Peptides regulate a plethora of physiological functions, mainly by interactions with specific cellular receptors, whereby they induce cellular signalling events, such as neurotransmission and release of hormones. Endogenous peptides have been associated with challenges as therapeutics due to their limited in vivo stability and bioavailability. However, the high specificity and low toxicity combined with improved ability to selectively modify and improve therapeutic properties of peptides has increased the relevance of peptides in drug development.
In endocrinology, diseases are often caused by or associated with an imbalance of the level of peptide hormones, as seen in diseases such as diabetes and obesity. Notably, about half the peptide hormones in the endocrine and nervous systems are α-amidated in their C-terminal and the α-amide moiety is often crucial for biological activity and stability. Certain therapeutic peptides including peptide hormones involved in obesity and diabetes (e.g. peptide YY (PYY), pancreatic peptide (PP), α-calcitonin gene related peptide (α-CGRP), calcitonin (CT), and amylin) require an α-amide moiety in the C-terminal to obtain full biological activity.
The most widely used technologies for production of peptide therapeutics are microbial expression systems and chemical synthesis. While a peptide C-terminal amide is easily achieved by chemical synthesis, it is not readily introduced into recombinant peptides derived from microbial hosts, which lack an α-amidating enzymatic machinery. Therefore, the α-amide has to be introduced as a post translational modification.
Inteins are autocatalytic protein domains which are expressed in unicellular organisms with flanking protein sequences at both amino- and carboxy-termini. The amino- and carboxy-terminal sequences have been named exteins in keeping with the DNA nomenclature of exons and introns. A seemingly typical member of the emerging family of inteins is the GyrA gene product from Mycobacterium xenopi (Mxe GyrA). This is approximately 22 kDa in molecular mass and contains a number of crucial amino acids at the amino-terminus (cysteine) and at the carboxy-terminus (histidine and asparagine). In addition, the carboxy-terminal extein must start with a cysteine, serine or threonine. At some point after translation is completed, the peptide bond between the amino-terminal extein and the intein is converted into a thioester bond by an N-to-S acyl shift involving the cysteine at the amino-terminal of the intein. This bond is then exchanged with the nucleophilic residue (serine, threonine or cysteine) at the start of the carboxy-terminal extein and then, with participation of the asparagine at the C-terminus of the intein, the intein excises itself out, while a second acyl shift generates a native peptide bond between the amino- and carboxy-terminal exteins. The overall effect of these concerted reactions is that the two exteins are seamlessly joined and the intein is released.
Mutant inteins have been designed where the self-splicing function has been disabled by a mutation to allow cleavage at either the amino- or carboxy-terminal splice junctions. For the Mxe GyrA intein, amino-terminal cleavage has been enabled by a N198A mutation. Replacement of the amino-terminal extein by another polypeptide sequence, the target peptide, enables preparation of the target peptide with a reactive carboxy-terminal α-thioester handle after cleavage of the resulting fusion protein with a nucleophilic chemical agent such as sodium 2-mercaptoethanesulfonate (MESNa). Such intein-derived α-thioesters can be reacted with any nucleophile and is useful as a chemical handle for chemical ligation, bioconjugation or amidation. The intein-based approach has been used to generate α-amidated peptides recombinantly in a laboratory scale (WO 98/50563 A1; WO 00/00625 A1; Cottingham I. R. et al., Nat. Biotechnol. 2001, 19, 974-977).
The primary limitation of using this technology for large scale production of C-terminally α-amidated peptides is the low yields generally observed, which may be ascribed to a combination of the large size of the intein and hydrolytic instability of the intein fusion protein. Introduction of a T3C mutation in the Mxe GyrA intein has been shown to be associated with reduced premature cleavage (Cui C. et al. Protein Expr. Purif. 2006, 50, 74-81). Furthermore, the size of the intein is large relative to that of the peptide hormones and a reduction in intein size could potentially improve the final yield of the peptide hormone by a more economical usage of the host protein synthesis machinery.